The interrelationship, in tumor rejection, between (1) lymphokine production, (2) T-cell receptor usage, and (3) cytotoxicity, as measured in perforin transgenic mice, will be investigated in this proposal. An immunological response to a normally nonimmunogenic tumor is enforced by lymphokinic transfection of the tumor cell prior to transplantation into mice. IL2 transfection of the non-immunogenic Lewis lung cell carcinoma (LLC) renders the tumor immunogenic in such a way that it is rejected in normal mice but not in immunodeficient mice lacking T-cells (Nude,SCID) or NK-cells (Beige). Hence, both a T-cell and an NK-cell response are required for tumor rejection of this particular tumor producing IL2 (LLC- IL2). Even though IL2 producing LLC can retard the growth of normal LLC, a complete immunity resulting in rejection of the non-lymphokine producing tumor has not been achieved. By transfecting LLC with various lymphokines singly, or in a new double expression vector, with two lymphokine cDNAs simultaneously, the induction of an immune response giving complete tumor immunity is sought. Combination of lymphokines will focus on IL2 with IL4, IL6, I18 or gamma interferon. Studies with singly transfected LLC gave rise only to partial immunity. In rejected or retarded tumors the T- cell response will be determined by measuring the presence of T-cell receptor alpha and beta v-region gene families present in tumor infiltrating T-cells. Using a set of already tested (in NOD mice) v- region gene primers for both TCR-chains and the corresponding c-region primer, the cDNAs of the TCRs, generated by reverse transcription, will be amplified by PCR and cloned. The sequence of the most predominant TCR families, present at the tumor site under the influence of various tumor produced lymphokines, will be established for the v-, D-, and J-segments. To manipulate the T-cell response and uncover rejecting or suppressing T- cell clones, mice will be immunized with synthetic peptides corresponding to the TCR sequence. The ensuing anti idiotype antibody and cellular response will be evaluated with regard to its effect on tumor rejection. These studies will be complemented by the isolation and cloning of T-cells specific for lymphokine producing and non-producing LLC. The role of cytotoxicity, specifically the role of perforin, in tumor rejection in this model will be studied by comparing the tumor response of perforin deficient transgenic mice to that of perforin sufficient mice. Perforin deficiency has been achieved in embryonal stem cells by homologous recombination and perforin deficient chimeras are currently under study. These studies will be complemented by reconstituting perforin deficient mice, as well as normal mice, with human perforin transgenes. Human perforin, detectable by antibody and Northern analysis and distinguishable from mouse perforin, is expressed under the murine perforin promoter and under various promoter deletions, designed to enhance perforin expression in T-cell subsets. Improved perforin expression in transgenic mice will be analyzed with regard to its effect on tumor rejection.